Fuel ethanol produced from renewable resources is one of the long-term solutions to global fossil fuel shortages, rising energy costs, and global warming effects related to increased atmospheric carbon dioxide. Fuel ethanol from renewable resources is produced by fermentation of sugars. Currently in the United States, glucose derived from corn grain is the most abundant sugar source for ethanol production. Due to the demands for corn grain as a feed and food supply, methods of converting various types of cellulosic biomass (including hemicellulose) to fermentable sugars are being developed. Sugar derived from this biomass source is a mixture of hexoses and pentoses, primarily glucose and xylose. As a result of developments in cellulosic biomass processing, these sugars may be released in high concentrations and used in fermentation in high concentrations to produce ethanol, with reduced water consumption and higher throughput. As such, conversion of biomass to ethanol poses great possibility for improving environmental impacts compared to fossil fuel ethanol production. Further, it provides a potentially economically viable alternative to fossil fuel ethanol production.
In addition to improvements in biomass processing, genetic engineering has been used to make improvements in microorganisms that are able to produce ethanol. In order to enhance the utilization of sugars from cellulosic biomass, the ethanologen Zymomonas (i.e., Z. mobilis) has been made capable of utilizing xylose by engineering strains for expression of four enzymes: 1) xylose isomerase, which catalyses the conversion of xylose to xylulose; 2) xylulokinase, which phosphorylates xylulose to form xylulose 5-phosphate; 3) transketolase; and 4) transaldolase (U.S. Pat. Nos. 5,514,583, 6,566,107; Zhang et al. (1995) Science 267:240-243). Though these strains do metabolize xylose, at high xylose concentration the xylose is not fully utilized, so that the theoretical ethanol yield is not achieved. The ethanol yield also is limited due to synthesis of xylitol as a by-product of xylose metabolism (Feldmann et al. (1992) Appl Microbiol Biotechnol 38: 354-361; Kim et al. (2000) Applied and Environmental Microbiology 66:186-193). Xylitol is toxic to cells due to its phosphorylation to xylitol 5-phosphate, which is a compound that accumulates in the cell and inhibits growth. The yield of ethanol is also reduced due to the synthesis of xylitol, since xylose-utilizing recombinant strains of Z. mobilis cannot convert xylitol to ethanol. In addition, xylitol is a potent inhibitor of xylose isomerase, which catalyzes the first step of xylose utilization in the engineered xylose metabolism pathway. Therefore, fermentations in high sugar medium including xylose, with xylose-utilizing Z. mobilis, do not achieve maximal xylose usage and ethanol production.
Complete use of 8% xylose in a sugars mixture with 4% glucose, by xylose utilizing Z. mobilis, took 2-3 days (Lawford and Rousseau (1999) Appl Biochem and Biotech. 77-79: 235-249). Complete use of 65 g/L xylose in a mixture with 65 g/L glucose required 48 hours (Joachimsthal and Rogers (2000) Appl Biochem and Biotechnol 84-86: 343-356), and using higher concentrations of sugars (75 g/L xylose and 75 g/L glucose) resulted in incomplete xylose utilization.
Sorbitol has been added as an osmoprotectant to enhance growth of non-engineered Z. mobilis in high concentrations of glucose (Loos et al. (1994) J Bacteriol 176:7688-7693). Sorbitol was accumulated intracellularly. In addition, sorbitol was shown to be produced by the cells and accumulated when growing on high sucrose. Any effects of sorbitol on production of ethanol by Z. mobilis strains that are engineered to utilize xylose, when grown in the presence of a sugar mixture including xylose, have not been determined previously.
There remains a need to develop fermentation conditions that enhance ethanol production in sugar media including xylose, allowing xylose-utilizing strains to reach their maximal ethanol production capacity with maximal xylose utilization in reduced time.